Transient Diffusive Interactions with a Protein Crowder Affect Aggregation Processes of Superoxide Dismutase 1 β-Barrel

Abstract

Aggregate formation of superoxide dismutase 1 (SOD1) inside motor neurons is known as a major factor in onset of amyotrophic lateral sclerosis. The thermodynamic stability of the SOD1 β-barrel has been shown to decrease in crowded environments such as inside a cell, but it remains unclear how the thermodynamics of crowding-induced protein destabilization relate to SOD1 aggregation. Here we have examined the effects of a protein crowder, lysozyme, on fibril aggregate formation of the SOD1 β-barrel. We found that aggregate formation of SOD1 is decelerated even in mildly crowded solutions. Intriguingly, transient diffusive interactions with lysozyme do not significantly affect the static structure of the SOD1 β-barrel but stabilize an alternative excited "invisible" state. The net effect of crowding is to favor species off the aggregation pathway, thereby explaining the decelerated aggregation in the crowded environment. Our observations suggest that the intracellular environment may have a similar negative (inhibitory) effect on fibril formation of other amyloidogenic proteins in living cells. Deciphering how crowded intracellular environments affect aggregation and fibril formation of such disease-associated proteins will probably become central in understanding the exact role of aggregation in the etiology of these enigmatic diseases.

In-cell NMR: Why and how?

“…More recently, Iwakawa et al studied the effect of a negatively charged crowding agent, lysozyme, on SOD1 b-barrel structure and oligomerization. Lysozyme slowed down the protein oligomerization, while NMR analysis of the monomer revealed that the interactions between lysozyme and SOD1 stabilized the latter in alternative excited state [1368].…”

Computational Insights into the Unfolding of a Destabilized Superoxide Dismutase 1 Mutant

“…The cleft between 5 and 6 was reported to become partially unstructured in excited states of certain destabilized mutants of the full-length apoSOD1 monomer, which might potentially lead to nonnative oligomerization [ 7 ]. In addition, some of these conformations might be related to a compact excited state of SOD1 which was detected by NMR experiments [ 13 ] and found to be stabilized by transient interactions with a protein crowder, leading to a slow-down of SOD1 aggregation [ 16 ].…”

“…The loop truncation entropically stabilizes SOD1 relative to the full-length disulfide-reduced apoSOD1 monomer [ 12 ]. To bring the stability of SOD1 closer to that of the disulfide-reduced apoSOD1 monomer, the isoleucine 35 to alanine (I35A) mutation is often introduced to the barrel [ 9 , 16 , 17 ], substantially decreasing the thermal stability of SOD1 and causing a large part of SOD1 molecules to be unfolded in the cell at 37 degrees Celsius [ 9 ]. Thus, although the I35A mutation is currently not known to cause familial ALS, the SOD1 mutant constitutes a convenient model system to investigate the effects of the cellular environment on the folding state of the destabilized apoSOD1 monomer.…”

Virtual Issue on Protein Crowding and Stability

“…26 Another NMR study found reduced aggregation of SOD1 in the presence of protein crowders. 27 This was interpreted to result from SOD1− crowder interactions that were competing with SOD1 aggregation as one example of crowding-induced effects on aggregation covered more generally in a theoretical analysis. 28 Homing in on biological function, a FRET-based analysis contrasts binding of Hsp70 and Hsc70 to a model protein substrate in living cells in the cellular background of other functional interactions of Hsc70.…”

“…Direct evidence of how the binding energetics of a peptide to an SH3 domain is modulated by the presence of different protein crowders or osmolytes comes from an NMR study . Another NMR study found reduced aggregation of SOD1 in the presence of protein crowders . This was interpreted to result from SOD1–crowder interactions that were competing with SOD1 aggregation as one example of crowding-induced effects on aggregation covered more generally in a theoretical analysis .…”